Proteinuria and TPR alteration were not related with significant baseline immunosuppresion (P = 0.61 and P = 0.73, respectively) nor low BMI (P = 0.60 and P = 0.30, respectively).
Patients who additionally received LPV/RTV or ddI did not exhibit more pronounced CrCl decreases or higher serum creatinine levels. There was, however, a statistically significant urine osmolality alteration in patients receiving LPV/RTV together with TDF (P = 0.027 and RR 8.7, 1.4–54). All patients receiving LPV/RTV or ddI showed moderate to severe proteinuria, as well as a lower TPR (Fig. 2).
The use of TDF is not unusual in the pediatric age, particularly in the adolescent population. Currently in Spain, 14% of patients in the CoRISpe cohort have received TDF at some time point as a part of their HAART regimens (unpublished data). The demographic and clinical characteristics of the patients included in our study are similar to those of the CoRISpe patients who received TDF, as well as to those included in previous studies [2,4,8], that is, mainly, adolescents who were given TDF as part of a salvage therapy. Thus, it is likely that the study results would be applicable to the overall population of pediatric patients receiving this drug. The findings of considerable tubular dysfunction observed in our patients do not differ from the results of other studies in children assessing renal toxicity associated with TDF use [2,8,25].
It has been well demonstrated that the specimen of choice to determine the existence of tubular proteinuria is 24-h urine [6,13,29], as we used in this study. Other, simpler methods, including urine reagent strips or the urinary protein/creatinine ratio, used by other authors  are less suitable for this purpose.
With regard to serum and urinary phosphate concentrations, TPR abnormalities were seen in 90% of patients and six of them showed a DAIDS grade 1 or 2 serum phosphate decrease. Moreover, serum phosphate alterations were significantly related to both TPR changes and time on TDF treatment. In the study by Judd et al. , a difference was seen in the incidence of hypophosphatemia (DAIDS grade 2) between patients on TDF and those who had never received this drug. Along this same line, Izzedine et al. suggested that hypophosphatemia (as well as glucosuria in the absence of hyperglycemia) would be a good marker of tubular toxicity due to TDF . In contrast, other authors contend that moderate, asymptomatic hypophosphatemia is normal in adult HIV-infected patients regardless of the HAART they receive [11,33]. Nonetheless, these studies were performed in adults and follow-up lasted less than 1 year, factors that could explain the differences with respect to our study that had a follow-up of around 6.5 years.
There were no cases of Fanconi syndrome in our series, an uncommon complication recently reported in patients receiving TDF [23,34].
It has been described that TDF-related renal toxicity may increase when the drug is combined with other antiretroviral drugs , particularly with LPV/RTV or ddI. Most patients in our cohort had been treated with several antiretroviral regimens, although few of them received TDF combined with LPV/RTV or ddI. The TDF–LPV/RTV combination was associated with moderate to severe proteinuria and TPR decrease, although the differences with respect to the remaining patients were not significant, possibly because of the small sample size. A significant association was found between urine osmolality alterations and combined use of TDF and LPV/RTV. This finding may be related to an increase in plasma TDF resulting from pharmacokinetic interactions between the two antiretroviral drugs [23,35], a factor that was not investigated in our study.
Considering that the peak in bone mineral density occurring in adolescence is inversely related with the risk of osteopenia, osteoporosis, and bone fracture in adulthood [36–37], the increased incidence of bone pathology in HIV-infected patients  supports the idea that monitoring calcium–phosphate metabolism in HIV-infected children and adolescents is of particular interest. Furthermore, some authors have found that TDF use is an independent risk factor for developing bone mineral loss . In an experimental animal study, Van Rompay et al.  demonstrated an association between prolonged, high-dose TDF administration and hypophosphatemia and development of osteomalacia.
The main limitation of our study is the relatively small sample size, although the comparability of the patients included with those receiving TDF in the CoRISpe cohort and other pediatric studies evaluating TDF-associated renal toxicity confers greater validity to the results obtained. In addition, because baseline evaluation of tubular function was not performed, it was not possible to establish a causal relationship in the results found. In any case, the documentation of significant proteinuria in a high percentage of patients and the correlation between phosphate levels and tubular dysfunction, as well as the optimum sample studied (24-h urine), lead us to suggest that TDF played an important role in the renal alterations described.
In conclusion, this is the first study prospectively assessing renal function in depth, including analysis of tubular function, in pediatric patients undergoing long-term TDF treatment. On the basis of the results obtained, we believe that periodic monitoring of tubular function should be included in the routine follow-up of these patients. Further investigation is needed to determine the contribution of TDF to alterations in calcium–phosphate metabolism and its potential association with bone metabolism alterations.
This study was funded in part by the AIDS Research Network (RIS, Red de Investigación en SIDA) and the Foundation for AIDS Research and Prevention in Spain (FIPSE, Fundación para la Investigación y la Prevención del SIDA en España; 240813/09).
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